Abstract

The Mycobacterium tuberculosis genome contains 11 serine/threonine kinase genes including two, pknA and pknB, that are part of an operon encoding genes involved in cell shape control and cell wall synthesis. Here we demonstrate that pknA and pknB are predominantly expressed during exponential growth, and that overexpression of these kinases slows growth and alters cell morphology. We determined the preferred substrate motifs of PknA and PknB, and identified three in vivo substrates of these kinases: PknB; Wag31, an ortholog of the cell division protein DivIVA; and Rv1422, a conserved protein of unknown function. Expression of different alleles of wag31 in vivo alters cell shape, in a manner dependent on the phosphoacceptor residue in the protein produced. Partial depletion of pknA or pknB results in narrow, elongated cells. These data indicate that signal transduction mediated by these kinases is a novel mechanism for the regulation of cell shape in mycobacteria, one that may be conserved among gram-positive bacteria.

Phenotype analysis of mycobacterial strains overexpressing pknA or pknB. Growth and viability of M. smegmatis (A) and M. bovis BCG (B) containing a single additional copy of pknA (filled gray circle), pknB (▵) pknAK2M (•), pknBK40M (▴) or plasmid only (▪), under the control of the acetamide-inducible promoter. Cultures were grown to early log phase, diluted to OD600 = 0.02 and cultured in the presence of inducer. Error bars are ±1 SD. In the bar graph, the pknB-overexpression strain is shown in white, the pknA-overexpression strain in gray, and the control in black. (C) Negative stain electron micrographs of M. smegmatis and M. bovis BCG cells containing a single additional copy of pknA, pknB, or plasmid under the control of the acetamide-inducible promoter in pMH94. Cultures were grown in the presence of inducer to early stationary phase, at which point they were harvested, fixed, and stained. Bar, 500 nm.

Auto- and cross-phosphorylation activity of PknA and PknB. Recombinant native and variant forms of the intracellular kinase domains of PknA and PknB were assayed in in vitro kinase reactions. Native proteins were purified as GST fusion proteins, and PknAK42M and PknBK40M mutant forms were 6X-His fusion proteins. Coomassie blue staining was performed to verify the amount of protein in each reaction.

Peptide library screening to determine the optimal substrate motif of PknA and PknB. (A) Library pool screening to determine a preferred library for PknA and PknB. The peptide libraries are named by the central fixed phosphoacceptor residue (S or T) and the position and identity of any other fixed amino acid in the library. Where not specified, all positions –4 to +4 relative to the phosphoacceptor are degenerate. Each degenerate peptide library was phosphorylated in the presence of [γ-32P]ATP by active PknA or PknB (black bars), or the inactive forms of these kinases (gray bars). (B) Time course of phosphorylation of a TQ versus an SQ library for each protein. (•) PknA + TQ peptide library; (▪) PknA + SQ peptide library; (♦) PknB + TQ peptide library; and (▴) PknB + SQ peptide library. Error bars are ±1 SD. (C) The TQ degenerate peptide library was incubated with active or inactive PknA or PknB followed by enrichment for phosphopeptides, as described in the Materials and Methods section. The phosphopeptide mixture was subject to quantitative Edman degradation, and the relative amount of each amino acid at each position was determined. Specific amino acids for which positive selectivity was observed are shown. Ratios are the amount of each amino acid from the peptides obtained following incubation with native kinase relative to that incubated with the inactive kinase. (D) In vitro phosphorylation of optimal and variant peptide substrates by PknA and PknB. In vitro phosphorylation reactions were performed in the presence of [γ-32P]ATP, and incorporation of 32P was determined. Error bars are ±1 SD.

Comparison by immunoblot analysis of phosphoprotein patterns in whole-cell lysates of M. tuberculosis cells harboring pMH94 vector alone, pMH94-pknA, or pMH94-pknB grown in the presence of inducer. Proteins were prepared from early stationary phase cultures after 24 h of induction, and subjected to 2DGE. Protein was then electrotransferred to PVDF membrane, followed by immunoblotting with a phospho-(S/T)Q-specific antibody and chemiluminescent detection. Arrowheads indicate the spots that showed stronger signals in gels from pknA- or pknB-overexpression cells than the control.

(A) In vitro phosphorylation of Rv1422 and Wag31 by PknA and PknB. Recombinant 6X-His-Rv1422, 6X-His-Rv1422T325A, 6X-His-Rv1422T325S, 6X-His-Wag31, 6X-His-Wag31T73A, or 6X-His-Wag31T73S was incubated with GST-PknA and/or GST-PknB in kinase buffer as described in the Materials and Methods section. After separation in SDS-PAGE gels, protein was electro-transferred to a PVDF membrane, with phosphoprotein detection using a phospho-(S/T)Q antibody and chemiluminescent detection. A 30-sec exposure was required to detect signals from the reaction with Wag31, while only 10 sec of exposure was used for Rv1422. (B) In vitro phosphorylation of synthetic Wag31 peptides by PknA and PknB. Kinase assays were performed in the presence of [γ-32P]ATP. Five micro-liters was taken at the indicated time points, and incorporation of 32P was determined. (○) GST-PknA + wild-type Wag31 peptide; (□) GST-PknA + Wag31T73A pep-tide; (⋄) GST-PknA + Wag31T73S peptide; (•) GST-PknB + wild-type Wag31 peptide; (▪) GST-PknB + Wag31T73A peptide; (♦) GST-PknB + Wag31T73S peptide.

Antisense RNA expression to inhibit PknA and PknB activity in vivo. M. smegmatis cells were grown to early exponential phase and expression of antisense pknA or pknB was induced. A strain expressing the xylE gene under control of the same promoter was used as a control. Cells were grown to late exponential phase and prepared for microscopic analysis or total protein extraction. (A) Growth curve determined by measurement of OD600. (▴) xylE-expressing strain; (•) antisense-pknA-expressing strain; (▪) antisense-pknB-expressing strain. (B–D) Negative stain electron microscopy of kinase antisense-expressing strains and control.

In vivo phosphorylation of Rv1422 and Wag31 and cell shape effects of wild-type and phosphoacceptor-site variants of Wag31 in M. smegmatis. (A) 2D immunoblots probed with a phospho-(S/T)Q-specific antibody of total protein from M. smegmatis cells harboring pMV306 plasmid alone, pMV306-Rv1422, or pMV306-wag31. Cells were grown to mid-log phase in the presence of 0.2% acetamide; total protein was extracted and subjected to 2DGE, immunoblotting, and chemiluminescent detection. For the two left panels, broad-range (pH 3–10) IEF strips were used; for the two panels on the right, narrow-range (pH 4.7–5.9) IEF strips were used to separate Wag31 from other (S/T)Q phosphorylated proteins. (B) Cell morphology phenotype of wag31 overexpression. Genes for M. tuberculosis Wag31, Wag31T73A, or Wag31T73E were expressed in M. smegmatis. Cells were cultured to early stationary phase without inducer, followed by addition of 0.03% acetamide, and grown for an additional 12 h prior to harvesting. All micrographs are at 15,000× magnification. Bar, 500 nm. The bar graph shows the percentage of short, wide cells in each strain, counted in two independent experiments. Error bars are ±1 SD. The immunoblot was performed with an anti-Wag31 monoclonal antibody. The upper band in each lane is the endogenous M. smegmatis Wag31, which is 20 amino acids larger than M. tuberculosis Wag31.